U.S. patent application number 10/052641 was filed with the patent office on 2003-02-13 for method for diagnosis and therapy of hodgkin's lymphomas.
Invention is credited to Beham-Schmid, Christine, Heider, Karl-Heinz, Zatloukal, Kurt.
Application Number | 20030032073 10/052641 |
Document ID | / |
Family ID | 7815732 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032073 |
Kind Code |
A1 |
Heider, Karl-Heinz ; et
al. |
February 13, 2003 |
Method for diagnosis and therapy of Hodgkin's lymphomas
Abstract
The invention relates to a method for diagnosing and treating
Hodgkin's lymphomas (lymphogranulomatosis) which is based on the
expression of the variant exon v10 of the CD44 gene as a molecular
marker or target. There is a significant correlation between v10
expression and the stage and prognosis of the disease. In a
preferred embodiment, v10-specific antibody molecules are used to
measure the expression of the exon in samples. In another preferred
embodiment, radiolabelled v10-specific antibodies are used to treat
Hodgkin's lymphomas.
Inventors: |
Heider, Karl-Heinz;
(Stockerau, AT) ; Zatloukal, Kurt; (Graz, AT)
; Beham-Schmid, Christine; (Graz, AT) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Family ID: |
7815732 |
Appl. No.: |
10/052641 |
Filed: |
January 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10052641 |
Jan 23, 2002 |
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09331254 |
Dec 21, 1999 |
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6372441 |
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09331254 |
Dec 21, 1999 |
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PCT/EP97/07081 |
Dec 17, 1997 |
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Current U.S.
Class: |
435/7.23 ;
424/155.1 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 33/6878 20130101; G01N 33/57492 20130101; C07K 16/2884
20130101 |
Class at
Publication: |
435/7.23 ;
424/155.1 |
International
Class: |
A61K 039/395; G01N
033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 1996 |
DE |
196 53 607.3 |
Claims
1. Method of diagnosing or treating Hodgkin's lymphomas
(lymphogranulomatosis), characterised in that this method is based
on the expression of the variable exon v10 of the gene CD44 as
molecular marker.
2. Method according to claim 1, characterised in that it is based
on the binding of an antibody molecule to an epitope which is coded
by the variable exon v10 of the gene CD44.
3. Method according to claim 2, characterised in that an antibody
molecule is used which recognises the amino acid sequence according
to SEQ ID NO. 2.
4. Method according to one of claims 1 to 3, characterised in that
the antibody molecule is a monoclonal antibody, an Fab- or
F(ab').sub.2-fragment of an immunoglobulin, a recombinantly
produced antibody, a recombinantly produced chimeric, humanised
antibody or single chain antibody (scFv).
5. Use of an antibody molecule which is specific to an epitope
which is coded by the variant exon v10 of the CD44 gene, in a
method according to one of claims 1 to 4.
6. Use of an antibody molecule which is specific for an epitope
within the amino acid sequence coded by the variable exon v10 of
the CD44 gene, for treating Hodgkin's lymphomas
(lymphogranulomatosis).
7. Use according to claim 6, characterised in that the antibody
molecule binds to the amino acid sequence according to SEQ ID NO.
2.
8. Use according to one of claims 6 and 7, characterised in that
the antibody molecule is a monoclonal antibody, a Fab- or
F(ab').sub.2-fragment of an immunoglobulin, a recombinantly
produced antibody, a recombinantly produced chimeric or humanised
antibody, or single chain antibody (scFv).
9. Use according to one of claims 6 to 8, characterised in that the
antibody molecule is linked to a radioactive isotope, a radioactive
compound, an enzyme, a toxin, a cytostatic, a prodrug, a cytokine
or another immunomodulatory polypeptide.
10. Use of an antibody molecule which is specific for an epitope
within the amino acid sequence coded by the variable exon v10 of
the CD44 gene, for preparing a pharmaceutical composition for
diagnosing and/or treating tumoral diseases.
11. Use according to claim 10, characterised in that the tumoral
disease is a Hodgkin's lymphoma (lymphogranulomatosis).
12. Use according to claim 10 or 11, characterised in that the
antibody molecule binds to the amino acid sequence according to SEQ
ID NO. 2.
13. Antibody molecule which is specific for an epitope within the
amino acid sequence coded by the variable exon v10 of the CD44
gene, for pharmaceutical use.
14. Antibody molecule according to claim 14, characterised in that
it binds to the amino acid sequence according to SEQ ID NO. 2.
15. Antibody molecule according to claim 13 or 14, characterised in
that it is a monoclonal antibody, a Fab- or F(ab').sub.2-fragment
of an immunoglobulin, a recombinantly produced antibody, a
recombinantly produced chimeric or humanised antibody or single
chain antibody (scFv).
16. Antibody molecule according to one of claims 13 to 15,
characterised in that it is linked to a radioactive isotope, a
radioactive compound, an enzyme, a toxin, a cytostatic, a prodrug,
a cytokine or another immunomodulatory polypeptide.
Description
[0001] The present invention relates to processes for diagnosing
and treating Hodgkin's lymphomas (lymphogranulomatosis) based on
the expression of the variant exon v10 of the gene CD44 as the
molecular target, agents for these processes and the use of these
agents.
[0002] The highly glycosylated cell surface protein CD44 is
involved in the interaction between cells and the extracellular
matrix such as migration and activation of leukocytes in
inflammation and immune monitoring, precursor formation of
leukocytic and myeloid cells in bone marrow and also in the
development of lymphoid organs and the interaction of cells with
the extracellular matrix (Lesley et al., 1993, Gunthert 1993, Pals
et al., 1993, Mackay et al., 1994). The human CD44 gene is made_up
of at least 19 exons, of which at least 12 which code for the
extracellular region are alternatively spliced (Screaton et al.,
1992). The CD44 gene is transcribed in a number of normal tissues
and carcinomas (Fox et al., 1994). Whereas the standard CD44
molecule (CD44s) is ubiquitously found expressed in epithelial and
mesenchymal tissues, the various isoforms produced by alternative
RNA splicing are found in very limited distribution (Heider et al.,
1993). Some of the variant isoforms are involved in the activation
of lymphocytes and occur in conjunction with metastasisation
(Mackay et al., 1994, Gunthert et al., 1991, Rudy et al., 1993,
Koopman et al., 1993). Although the expression of variant CD44 has
been shown to have a direct biological role in metastasis formation
in carcinoma of the pancreas in rats (Gunthert et al., 1991, Seiter
et al., 1993), its role in human tumours is as yet unknown.
[0003] Various reports have been published showing that certain
alternatively spliced forms of CD44 were expressed in human
metastatic tumours (Heider et al., 1993 and 1996, Fox et al., 1994,
Friedrichs et al., 1995, Kaufmann et al., 1995, Salles et al.,
1993, Stauder et al., 1995, Koopman et al., 1993, Tanabe et al.,
1993). Studies of the expression of CD44 in non-Hodgkin's lymphomas
(NHL) concentrated on analysing the so-called lymphocyte homing
receptor CD44H or CD44s (Horst et al., 1990a, Horst et al., 1990b,
Jalkanen et al., 1991, Moller et al., 1992). Whereas some authors
(Horst et al., 1990a, Jalkanen et al., 1991, Picker et al., 1988,
Pals et al., 1989, Fujiwara et al., 1993) found a correlation
between increased CD44s expression and unfavourable prognosis,
other authors (Terpe et al., 1994) could not confirm these
findings. Recently, upregulation of CD44v3 and CD44v6 isoforms was
found in NHL with unfavourable pathological status (Koopman et al.,
1993, Terpe et al., 1994, Salles et al., 1993, Stauder et al.,
1995), whilst variant specific CD44-mAbs were used (Mackay et al.,
1994, Koopman et al., 1993, Fox et al., 1993).
[0004] Various approaches have been developed for making use of the
differential expression of variant exons of the CD44 gene in
tumours and normal tissues for diagnostic and therapeutic purposes
(WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP
0531300).
[0005] The aim of the present invention was to develop new methods
of diagnosing and treating Hodgkin's lymphomas
(lymphogranulomatosis) and preparing agents for such processes.
[0006] This aim is achieved by means of the present invention. It
relates to processes for diagnosing and treating Hodgkin's
lymphomas (lymphogranulomatosis) which are based on the expression
of the variant exon V10 of the CD44 gene as a molecular marker or
target. Antibody molecules of corresponding specificity are
particularly suitable as vehicles for selectively reaching
Hodgkin's lymphomas in vivo.
[0007] Preferred processes are characterised in that an antibody
molecule is used which binds specifically to the amino acid
sequence SEQ ID NO. 2 (see Sequence Listing).
[0008] Other aspects of the present invention are the use of
antibody molecules of this kind in the processes according to the
invention and agents for performing these processes.
[0009] The invention further relates to the use of an antibody
molecule which is specific to an epitope within the amino acid
sequence which is coded by the variable exon V10 of the CD44 gene,
for preparing a pharmaceutical composition for the diagnosis and/or
treatment of tumoral diseases. The tumoral disease in question is
preferably Hodgkin's lymphoma (lymphogranulomatosis).
[0010] The invention further relates to an antibody molecule which
is specific to an epitope within the amino acid sequence which is
coded by the variable exon V10 of the CD44 gene for pharmaceutical
use. Preferably, an antibody molecule of this kind is characterised
in that it binds to SEQ ID NO. 2. It may be, in particular, a
monoclonal antibody, an Fab- or F(ab').sub.2-fragment of an
immunoglobulin, a recombinantly produced antibody, a recombinantly
produced chimeric or humanised antibody or single chain antibody
(scFv). Preferably, an antibody molecule of this kind is linked to
a radioactive isotope, a radioactive compound, an enzyme, a toxin,
a cytostatic, a prodrug, a cytokine or some other immunomodulatory
polypeptide.
[0011] The nucleic and amino acid sequence of the variant exon V10
of the CD44 gene is known (Screaton et al., 1992, Tolg et al.,
1993). These sequences are shown in the Sequence Listing (SEQ ID
NO. 1 and 2). The existence of degenerate or allelic variants is
unimportant to the performance of the invention; such variants are
therefore expressly included.
[0012] The invention may be carried out with polyclonal or
monoclonal antibodies specific to an epitope which is coded by the
exon v10. The preparation of antibodies to known amino acid
sequences can be carried out using methods known per se (Catty,
1989). For example, a peptide of this sequence may be prepared
synthetically and used as an antigen in an immunisation procedure.
Another method is to prepare a fusion protein which contains the
desired amino acid sequence, by integrating a nucleic acid (which
may be prepared -synthetically or, for example, by polymerase chain
reaction (PCR) from a suitable probe) which codes for this
sequence, into an expression vector and expressing the fusion
protein a host organism. The fusion protein, optionally purified,
can then be used as an antigen in an immunisation procedure and
insert-specific antibodies or, in the case of monoclonal
antibodies, hybridomas which express insert-specific antibodies,
are selected by suitable methods. Such methods are known in the
art. Heider et al. (1993, 1996) and Koopman et al. (1993) describe
the preparation of antibodies against variant epitopes of CD44.
[0013] However, for the process according to the invention, it is
also possible to use antibody molecules derived from poly- or
monoclonal antibodies, e.g. Fab- or F(ab') .sub.2-fragments of
immunoglobulins, recombinantly produced single chain antibodies
(scFv), chimeric or humanised antibodies and other molecules which
bind specifically to epitopes coded by exon v10. From a complete
immunoglobulin it is possible for example to produce Fab-or
F(ab').sub.2-fragments or other fragments (Kreitman et al., 1993).
The skilled person is also capable of producing recombinant
v10-specific antibody molecules. Corresponding methods are known in
the art. Recombinant antibody molecules of this kind may, for
example, be humanised antibodies (Shin et al., 1989; Gussow and
Seemann, 1991), bispecific antibodies (Weiner et al., 1993;
Goodwin, 1989), single chain antibodies (scfv, Johnson and Bird,
1991), complete or fragmentary immunoglobulins (Coloma et al.,
1992; Nesbit et al., 1992; Barbas et al., 1992), or antibodies
produced by chain shuffling (winter et al., 1994). Humanised
antibodies may be produced for example by CDR grafting (EP
0239400). Framework regions may also be modified (EP 0519596). In
order to humanise antibodies, nowadays it is possible to use
methods such as PCR (cf. for example EP 0368684; EP 0438310; WO
9207075) or computer modelling (cf. for example WO 9222653). It is
also possible to prepare and use fusion proteins such as single
chain antibody/toxin fusion proteins (Chaudhary et al..sub., 1990;
Friedman et al., 1993). The headings "antibody" and "antibody
molecules" should include, in addition to polyclonal and monoclonal
antibodies, all the compounds discussed in this section as well as
other compounds which are structurally derived from immunoglobulins
and can be prepared by methods known per se.
[0014] For diagnostic purposes, antibody molecules may be linked,
for example, to radioactive isotopes such as .sup.131I, .sup.111In,
.sup.99mTc or radioactive compounds (Larson et al., 1991; Thomas et
al., 1989; Srivastava, 1988), enzymes such as peroxidase or
alkaline phosphatase (Catty and Raykundalia, 1989), with
fluorescent dyes (Johnson, 1989) or biotin molecules (Guesdon et
al., 1979). For therapeutic applications, v10-specific antibody
molecules may be linked to radioisotopes such as .sup.90Y,
.sup.111In, .sup.131I or .sup.186Re (Quadri et al., 1993; Lenhard
et al., 1985, Vriesendorp et al., 1991; Wilbur et al., 1989) toxins
(Vitetta et al., 1991; Vitetta and Thorpe, 1991; Kreitman et al.,
1993; Theuer et al., 1993) cytostatics (Schrappe et al., 1992),
prodrugs (Wang et al., 1992; Senter et al., 1989) or radioactive
compounds. The antibody may also be linked to a cytokine or another
immunomodulatory polypeptide, e.g. tumour necrosis factor or
interleukin-2.
[0015] Advantageously, the diagnostic process according to the
invention can be used to examine samples from patients, e.g. from
biopsies, where there is a suspicion of Hodgkin's lymphoma
(lymphogranulomatosis) or where this has already been diagnosed but
the tumour requires more accurate characterisation. Variant CD44
molecules which contain an amino acid sequence coded by the
variable exon v10 can be detected at the protein level by means of
antibodies or at the nucleic acid level by means of specific
nucleic acid probes or primers for polymerase chain reaction (PCR).
The invention consequently also relates to antibody molecules and
nucleic acids which are suitable as probes or primers for such
processes, and the use of these antibodies and nucleic acids for
the diagnosis and analysis of Hodgkin's lymphomas. For example,
tissue sections can be investigated immunohistochemically with
antibodies using methods known per se. Extracts or body fluids
obtained from tissue samples can also be investigated by other
immunological methods using antibodies, e.g. by Western blots,
enzyme-linked immunosorbant assays (ELISA, Catty and Raykundalia,
1989), radioimmunoassays (RIA, Catty and Murphy, 1989) or related
immunoassays. The samples may be investigated qualitatively,
semiquantitatively or quantitatively. The expression of the
CD44-splice variant v10 in Hodgkin's disease is associated with
aggressive behaviour of the tumour and a high risk of recurrence.
This correlates with an advanced stage and poor prognosis of NSHD
(nodular sclerosis Hodgkin's disease)
[0016] As well as in vitro diagnosis, antibody molecules with
specificity according to the invention are also suitable for in
vivo diagnosis of Hodgkin's lymphomas. If the antibody molecule
carries a detectable label, the label can be detected for
diagnostic purposes, e.g. imaging the tumour in vivo or for
radioguided surgery, for example. For using antibodies conjugated
with radioactive isotopes for immunoscintigraphy (imaging), for
example, there are a number of procedures on the basis of which the
skilled person can perform the invention (Siccardi et al., 1989;
Keenan et al., 1987; Perkins and Pimm, 1992; Colcher et al., 1987;
Thompson et al., 1984).
[0017] Data obtained by detecting and/or quantifying the expression
of the variant CD44 epitope v10 can thus be used for diagnosis and
prognosis. It may be advantageous to combine such data with other
prognostic parameters, e.g. with the grade of tumour.
[0018] Antibody molecules with the specificity according to the
invention and optionally linked with a cytotoxic agent may
advantageously be used to treat Hodgkin's lymphomas
(lymphogranulomatosis). They may be administered systemically or
topically, e.g. by intravenous route (as a bolus or continuous
infusion), or by intraperitoneal, intramuscular or subcutaneous
injection/infusion. Methods of administering conjugated or
non-conjugated antibodies, e.g. complete immunoglobulins,
fragments, recombinant humanised molecules etc.) are known in the
art (Mulshine et al., 1991; Larson et al., 1991; Vitetta and
Thorpe, 1991; Vitetta et al., 1991; Breitz et al., 1992, 1995;
Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989; Sears
et al., 1982).
[0019] The antibody molecules may be formulated in a manner known
per se. For example, they may be present in aqueous solution,
optionally buffered with a physiologically acceptable buffer A
solution of this kind may be characterised by the addition of
suitable stabilisers and adjuvants. However, the antibody molecules
may also be present in the form of a freeze-dried preparation
(lyophilisate) which is reconstituted with a suitable solvent, e.g.
water, before use.
[0020] In a preferred embodiment of therapeutic application, a
humanised v10-specific immunoglobulin or an F(ab').sub.2-fragment
thereof is linked with .sup.90Y (Quadri et al., 1993; Vriesendorp
et al., 1995), .sup.131I (Juweid et al., 1995; Press et al., 1995;
Thomas et al., in: Catty 1985, p. 230-239), .sup.186Re (Breitz et
al., 1992, 1995) or another suitable radioisotope and used for
radioimmunotherapy of Hodgkin's lymphomas. For example, an antibody
molecule of this kind may be linked with .sup.90Y using a chelating
linker such as ITCB-DTPA (isothiocyanatobenzyl-diethyle- netriamine
pentacetate), whilst a specific activity of 5-20 mCi/mg, preferably
10 mci/mg should be achieved. This agent can then be administered
to a patient with an antigen-positive tumour in a dosage of 0.1 to
1 mCi/kg of body weight, preferably 0.3 to 0.5 mCi/kg of body
weight, most preferably 0.4 mCi/kg. When the total quantity of
protein to be administered is from 2 to 5 mg this may be given in
the form of a rapid intravenous bolus injection. In the case of
monoclonal antibodies it may be necessary to mix the agent with an
excess (e.g. a ten-fold molar excess) of the non-radioactive
antibody before administering it; in this case, the preparation is
better administered in the form of an intravenous infusion over a
period of 15 minutes, for example. The application can be repeated.
The treatment can be backed up by bone marrow transplantation.
FIGURES
[0021] FIG. 1: A. Individual HRS (Hodgkin and Reed-Sternberg) cells
of a patient with no recurrence, reacting with mAb VFF16 (CD44v10).
The arrow tips point to non-reactive HRS cells. B. >50% of the
HRS cells of patients with a recurrence show reactivity
(ABC,.times.400).
[0022] FIG. 2: CD44V10 expression in HRS cells in various patient
groups. It should be noted that patients with a poor clinical
progress, i.e. recurrence or bone marrow involvement exclusively
show more than 10%. positive HRS cells, whereas patients with no
recurrence have less 10% positive HRS cells. The difference between
these two groups is statistically highly significant.
[0023] FIG. 3: RT-PCR analysis using CD44v10-specific primers
(right half): the main transcript of about 470 bp in all the probes
and a weaker transcript of 660 bp in probes 2, 3 and 4 indicate
CD44v10-containing isoforms in all 5 cases A dominant band of 440
bp when using primers which are specific to the 5'- and 3'-constant
region indicates the standard form of CD44 (left half).
EXAMPLES
Example 1
[0024] Preparation of v10-specific antibodies
[0025] The entire variant region of the HPKII type of CD44v
(Hofmann et al., 1991) was amplified from human keratinocyte cDNA
by polymerase chain reaction (PCR). The two PCR primers
5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3', positions 25-52, and
5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3', positions 1013-984 of the
LCLC97-variant region as described by Hofmann et al. contained an
EcoRI recognition site which was used to clone the PCR product
directly into the vector pGEX-2T (Smith et al., 1988). The
resulting construct (PGEX CD44v HPKII, v3-v10) codes for a fusion
protein of .about.70 kD, consisting of glutathione-s-transferase
from Schistosoma japonicum and the exons v3-v10from human CD44
(Heider et al., 1993). The fusion protein was expressed in E. coli
and then affinity-purified over glutathione-agarose (Smith et al.,
1988).
[0026] Female Balb/c mice were immunised by intraperitoneal route
with the affinity-purified fusion protein according to the
following plan:
[0027] 1.sup.st immunisation: 90 .mu.g of fusion protein in
complete Freund's adjuvant
[0028] 2.sup.nd and .sub.3.sup.rd immunisations: 50 .mu.g of fusion
protein in incomplete Freund's adjuvant.
[0029] The immunisations were carried out at intervals of 4 weeks.
14 days after the last immunisation, the animals were immunised on
three successive days with 10 .mu.g of fusion protein in PBS. The
next day, spleen cells from an animal with a high antibody titre
were fused with P3.X63-Ag8.653 murine myeloma cells using
polyethyleneglycol 4000. The hybridoma cells were then selected in
microtitre plates in HAT medium (Kohler and Milstein, 1975; Kearney
et al., 1979).
[0030] The antibody titre in the serum and the screening of the
hybridoma supernatants were carried out using an ELISA. In this
test, first of all, microtitre plates were coated with fusion
protein (GST-CD44v3-10) or with glutathione-S-transferase on its
own. Then they were incubated with serial dilutions of serum
samples or hybridoma supernatants and the specific antibodies were
detected with peroxidase-conjugated antibodies against murine
immunoglobulin. Hybridomas which reacted only with
glutathione-S-transferase were discarded. The remaining antibodies
were characterised first in an ELISA with domain-specific fusion
proteins (exon v3, exon v5+v6, exon v6 +v7, exon v8-v10, exon v10)
(Koopman et al., 1993). Their immunohistochemical reactivity was
tested on sections of human skin.
[0031] Antibodies from the supernatants of the hybridoma clones
VFF-14 and VFF-16 bind only to fusion proteins containing a domain
which is coded by the exon v10.
Example 2
[0032] Immunohistochemical examination of tissue samples
[0033] Tissue and patients
[0034] 37 Paraffin-embedded lymph node samples from 29 patients
with NSHD (nodular sclerosis Hodgkin's disease; according to Rye
classification) were obtained from the collection of the Pathology
Department, University Medical School, Graz, Austria, and divided
into three groups; group 1: 11 patients with pretreated NSHD before
treatment (5 patients at stage I, 6 at stage II), who had been free
from recurrence for more than 6 years; group 2: 9 patients with
pretreated NSHD before treatment (4 at stage I, 5 at stage II)
suffered a recurrence of the disease in one to three years. Two to
three follow-up lymph node sections of NSHD recurrences from 7 of
these 9 patients were also included in this study; group 3: 9
patients with bone marrow involvement at the time of the original
diagnosis (stage IV).
[0035] Immunohistochemistry
[0036] The lymph node samples were stained with the following mAbs:
CD44 standard (s) recognised by the mAb SFF2; CD44v5 detected by
the mAb VFF8; CD44v6 detected by the mAbs VFF7 and VFF18; CD44v10
detected by the mAbs VFF14 and VFF16. Mab SFF2 recognises an
epitope common to all CD44-isoforms. Mabs VFF7 and VFF18 recognise
different but overlapping epitopes which are coded by the exon v6.
Mab VFF8 is specific to exon v5. Mabs VFF14 and VFF16 react with an
epitope which is coded by exon v10.
[0037] The immunohistochemistry was carried out on sections treated
with microwaves (Gerdes et al., 1992), using the avidin-biotin
complex (ABC) peroxidase method (Guesdon et al., 1979). Paraffin
sections were dewaxed in xylene, rehydrated and the endogenous
peroxidase was blocked with H.sub.2O.sub.2 in methanol. The slides
were placed in a glass stand and wetted in 500 ml of 0.01 M citrate
buffer (2.1 g of citric acid in 1 litre of deionised water, pH
adjusted to 6.0 using 2 N NaOH). The microwave treatment was
carried out for 35 hours at maximum power (600 W) in a microwave
oven (BioRad). After 9 minutes' microwave treatment the evaporated
buffer was topped up with deionised water. After the microwave
irradiation the solution was cooled for 20 minutes. Then the slides
were rinsed in phosphate-buffered saline (PBS) and immunostained by
diaminobenzidine (DAB) development.
[0038] For comparison, 10 frozen lymph node samples (3 from
patients of group 1, 2 from group 2 and 5 recently collected cases)
were also incubated with the mAbs VFF14 (v10) and VFF16 (v10),
using the alkaline phosphatase-anti-alkaline phosphatase (APAAP)
method (Cordell et al., 1984).
[0039] For control purposes, sections of normal human epidermis
which is known to contain the antigens in question were tested
(positive controls). Replacement of the primary antibody by normal
serum always produced negative results (negative controls).
[0040] As an additional control, immunohistochemical staining for
CD44v10and CD44v6 expression was repeated twice in each case. To
confirm the findings, the cases were additionally incubated in a
different laboratory using the APAAP method (Cordell et al.,
1984).
[0041] The percentage of HRS (Hodgkin and Reed-Sternberg) cells
stained with the antibodies was graded as 0, less than 10%, 10-50%
and over 50%. Care was taken to ensure that the immunoreactive HRS
cells were clearly tumour cells (e.g. by looking for the presence
of characteristic nuclear details), particularly in those cases
where less than 10% of the HRS cells expressed the antigens in
question. All the cases were assessed separately by two of the
inventors. The spread of the staining, e.g. on the membrane, in the
cytoplasm or both, was recorded, as well as the immunoreactivity in
cells which were not HRS cells.
[0042] Statistical analysis
[0043] CD44 expression patterns were analysed using the
Pearson-chi-square calculation and the Mantel-Haenszel test for
linear association, by means of the program SPSS for Windows.
P-values less than or equal to 0.05 were regarded as
significant.
[0044] Results of the immunohistochemical staining
[0045] Table 1 shows a summary of the results obtained with
antibodies directed against CD44s, CD44v5, v6 and v10 in HRS cells.
The majority of the antigenic reactivity of the HRS cells was on
the cell surface in every case. A variable number of HRS cells
yielded cytoplasmic and/or dot-like perinuclear reactivity with or
without surface staining, which probably reflected the reactivity
of CD44 molecules in the Golgi apparatus or in the endoplasmic
reticulum.
[0046] CD44v10 expression correlates with an advanced stage and
poor prognosis of NSHD. CD44s-, CD44v5-(detected by mAb VFF8) and
CD44v6-expression. (detected by mAb VEF18) in HRS cells was found
in the majority of cases, although there was great variation in the
number of stained cells (Table 1). CD44v6 expression (detected by
mAb VFF7) was found in only a few cases and, when present, was
restricted to a minority of HRS cells (Table 1). In patients with
no recurrence, CD44v10 expression (detected by the mAbs VFF14 and
VFF16) was found in only a very few cases, and the proportion of
reactive HRS cells was <10% (FIGS. 1A and 2). By contrast, in
every case of a patient with a recurrence or initial involvement of
the bone marrow, CD44v10 expression was found In the majority of
these cases, there was a clear overexpression of CD44v10 with
>50% reactive HRS cells (FIGS. 1B and 2). This overexpression of
CD44v10 was also found in the lymph node sections of recurrences
studied (Table 2).
[0047] Frozen lymph node sections from 3 patients with no
recurrence and from 2 patients with a recurrence yielded exactly
the same results as paraffin sections. The two antibodies specific
to exon v10 (VFF14 and VFF16) showed identical results in the
reaction either on the surface and/or in the cytoplasm of the HRS
cells.
[0048] Using Fisher's exact test, the differences in the CD44
isoform expression between groups of patients with non-aggressive
and aggressive NSHD yielded the following p-values: CD44s
(p=0.3625), CD44v5 (p=0.2415), CD44vG (detected with mAb VFF7)
(p=0.2903), CD44v6 (detected by mAb VFF18) (p=0.1836), CD44v10
(detected by mAbs VFF14 and VFF16) (p=0.001). This shows that the
different expression patterns of CD44v10 (detected by mAbs VFF14
and VFF16) within these NSHD groups were statistically highly
significant. The expression of the CD44 splice variant v10 in
Hodgkin's disease is associated with aggressive behaviour of the
tumour and a high risk of recurrence. It correlates with an.
advanced stage and poor prognosis of NSHD.
[0049] By contrast with earlier immunohistochemical studies of CD44
expression in conjunction with prognostic relevance in neoplasia of
different histogenetic origin, which were carried out exclusively
on frozen sections (Koopman et al., 1993, Terpe et al., 1994;
Ristamaki et al., 1994, 1995, Stauder et al., 1994, Heider et al.,
1993, Horst et al., 1990a,b, Heider et al., 1996, Kaufmann et al.,
1995, Mulder et al. 1994), the present invention was able to
demonstrate that CD44-mAbs can also be used on paraffin-embedded
material if microwave treatment is used. This process requires
constant fixing and microwave treatment in order to give
reproducible results. In order to validate the immunoreactivity
obtained with paraffin-embedded material, the immunohistochemical
analysis was carried out in parallel on frozen samples and
identical results were obtained. In addition, the results of the
CD44v10 expression were confirmed by RT-PCR. For the CD44v6
expression, a correlation could be demonstrated with poor prognosis
in NHL (Koopman et al., 1993, Salles et al., 1993, Terpe et al.,
1994, Ristamki et al., 1994, Stauder et al., 1994), breast cancer
(Kaufmann et al., 1995) and colon carcinomas (Heider et al., 1993).
In the cases of NSHD which we investigated, however, only a few
cases were CD44v6-positive and we could not detect any correlation
with the prognosis, using two different antibodies against CD44v6.
Because these two mAbs used against v6 recognise different epitopes
of the exon v6-coded amino acid sequence, this absence of
detectable CD44v6 in the majority of cases (see Table 1) cannot be
explained by modification or masking of epitopes. By contrast with
the frequent expression of CD44v5 in gastric adenocarcinomas
(Heider et al., 1993) the data relating to CD44v5 expression within
the three groups of NSHD were not statistically significant.
[0050] Exon v10, in addition to exons v3 and v6, is a variant exon
which is constitutively expressed in lymphocytes (Stauder et al.,
1994). Up till now, CD44v10 expression in NHLs has not been
systematically analysed and this exon has only rarely been detected
in carcinomas (Heider et al., 1996). The present invention
demonstrates, by the example of two different antibodies against
CD44v10 (VFF14 and VFF16), a statistically significant high
regulation of CD44v10 expression in HRS cells of NSHD with a poor
prognosis (groups 2 and 3). Both exon v10-specific antibodies
showed identical results, both on the surface and also (and/or) in
the cytoplasm of the HRS cells. The detection of CD44v10 expression
with two different mAbs is important because, for example, in
breast cancer different data were obtained by different authors
using different mAbs of the same exon specificity (Friedrichs et
al., 1995, Kaufmann et al., 1995). To confirm our surprising
results still further, all the cases were independently
immunostained in a different laboratory (using a different staining
method), with identical results.
[0051] The results for the CD44v10 expression in HRS cells of NSHD
are the first data which show a correlation between CD44v10
expression and the stage and prognosis of the disease. The methods
according to the invention thus provide the doctor with valuable
diagnostic and prognostic information on Hodgkin's lymphoma.
Moreover, CD44v10 is a suitable molecular target for therapeutic
interventions in this disease.
1TABLE 1 Reactivity of HRS cells CD44s v5 v6 v6 v10 v10 SFF2 VFF8
VFF7 VFF18 VFF14 VFF16 Patients % n % n % n % n % n % n % Group 1 n
= 11 >50 3 27.3 0 0 0 0 1 9.1 0 0 0 0 10-50 3 27.3 1 9.0 0 0 0 0
0 0 0 0 <10 3 27.3 6 54.5 2 18.2 8 72.7 4 36.4 4 36.4 0 2 18.1 4
36.5 9 81.8 2 18.2 7 63.6 7 63.6 Group 2 n = 9 >50 2 22.2 1 11.1
0 0 1 11.0 4 44.4 4 44.4 10-50 5 55.6 1 11.1 1 11.1 4 44.5 5 55.6 5
55.6 <10 2 22.2 7 77.8 3 33.3 4 44.5 0 0 0 0 0 0 0 0 0 5 55.6 0
0 0 0 0 0 Group 3 n = 9 >50 0 0 0 0 0 0 0 0 5 55.6 5 55.6 10-50
6 66.7 1 11.1 1 11.1 2 22.2 4 44.4 4 44.4 <10 3 33.3 8 88.9 0 0
7 77.8 0 0 0 0 0 0 0 0 0 8 88.9 0 0 0 0 0 0
[0052]
2TABLE 2 Reactivity (%) of CD44v10 (VFF14 and VFF16) and CD44v6
(VFF18) in HRS cells of patients of group 2 (recurrence) CD44v10
CD44v10 CD44v6 Patients (VFF14) (VFF16) (VFF18) 1 >50 >50
10-50 1 recurrence 10-50 10-50 10-50 1 recurrence 10-50 10-50 10-50
2 >50 >50 10-50 2 recurrence 10-50 10-50 10-50 3 10-50 10-50
<10 3 recurrence 10-50 10-50 <10 4 10-50 10-50 <10 4
recurrence 10-50 10-50 >50 5 10-50 10-50 10-50 5 recurrence
10-50 10-50 10-50 6 >50 >50 <10 6 recurrence >50 >50
10-50 7 >50 >50 <10 7 recurrence 10-50 10-50 <10 8
10-50 10-50 10-50 9 10-50 10-50 >50
Example 3
[0053] Use of v10-specific RT-PCR for diagnostic purposes
[0054] Reverse transcription polymerase chain reaction (PCR)
[0055] Five cases enabled mRNA to be isolated and were additionally
analysed by reverse transcriptase polymerase chain reaction
(RT-PCR).
[0056] 1 .mu.g of total RNA was isolated and reverse transcribed as
described in the literature (Gunthert et al., 1991). 5 .mu.l of
first strand cDNA were amplified with Taq polymerase (Promega,
Madison, USA) in a volume of 50 .mu.l, using the buffer conditions
recommended by the manufacturer. The concentration of primer was
0.2 mM. In order to test the quality and frequency of cDNA
synthesis, a GAPDH-PCR was carried out with oligonucleotides which
were homologous to positions 8-29 and 362-339 of the published
GAPDH-cDNA sequence (Allen et al., 1987). Pre-incubation for 5
minutes at 95.degree. C. was followed by 25 amplification cycles
(30 seconds at 95.degree. C., 1.5 minutes at 62.degree. C.) and an
extension cycle of 7 minutes at 72.degree. C. Then 10 .mu.l of the
reaction were analysed on a 2% agarose gel and the amplification
product was inspected under UV light after the gel had been stained
with ethidium bromide. In order to amplify cDNAs containing
CD44vlO, primers were used which were homologous with the 3'-end of
exon v10 (positions 986-1013, Hofmann et al., 1991) and with the
5'-constant region of CD44 (positions 513-540, Stamenkovic et al.,
1989). In order to amplify isoforms containing CD44 standard, a
3'-constant CD44 primer (positions 934-958, Stamenkovic et al.,
1989) was used instead of the CD44v10-specific primer. After 40
amplification cycles (94.degree. C. for 30 seconds, 62.degree. C.
for 1.5 minutes), 10 .mu.l of the reaction mixture were analysed as
above. For control purposes, instead of RNA, either distilled water
was used (negative control) or a plasmid containing CD44v3-v10 was
used (positive control, Heider et al., 1996).
[0057] In the 5 cases in which it was possible to isolate RNA, the
RT-PCR analysis confirmed the expression of CD44v10 containing
CD44-isoforms (as they were samples which had only recently been
obtained, the further progress of the disease in these patients is
not yet known). The amplified fragments correspond to CD44
transcripts which contain the constant proportion of CD44 combined
with the variant exon v10 (460 bp band) or variant exon v10 plus
other variant exons (660 bp band) (FIG. 3, right half). For control
purposes, parallel cDNAs were amplified with primers which were
specific for the 5'- and 3'-constant region of CD44 (FIG. 3, left
hand side), obtaining a prominent band of 440 bp which indicates
the standard form of CD44. The molecular-genetic results correlated
with the immunohistochemical findings, where in all 5 cases a high
proportion of the HRS cells (which represented less than 10% of the
total number of cells in a sample) expressed CD44v10 and the
majority of the cells (HRS plus non-tumour cells) reacted with the
anti-CD44s antibody.
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Sequence CWU 1
1
4 1 204 DNA Homo sapiens CDS (3)..(203) 1 at agg aat gat gtc aca
ggt gga aga aga gac cca aat cat tct gaa 47 Arg Asn Asp Val Thr Gly
Gly Arg Arg Asp Pro Asn His Ser Glu 1 5 10 15 ggc tca act act tta
ctg gaa ggt tat acc tct cat tac cca cac acg 95 Gly Ser Thr Thr Leu
Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr 20 25 30 aag gaa agc
agg acc ttc atc cca gtg acc tca gct aag act ggg tcc 143 Lys Glu Ser
Arg Thr Phe Ile Pro Val Thr Ser Ala Lys Thr Gly Ser 35 40 45 ttt
gga gtt act gca gtt act gtt gga gat tcc aac tct aat gtc aat 191 Phe
Gly Val Thr Ala Val Thr Val Gly Asp Ser Asn Ser Asn Val Asn 50 55
60 cgt tcc tta tca g 204 Arg Ser Leu Ser 65 2 67 PRT Homo sapiens 2
Arg Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn His Ser Glu Gly 1 5
10 15 Ser Thr Thr Leu Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr
Lys 20 25 30 Glu Ser Arg Thr Phe Ile Pro Val Thr Ser Ala Lys Thr
Gly Ser Phe 35 40 45 Gly Val Thr Ala Val Thr Val Gly Asp Ser Asn
Ser Asn Val Asn Arg 50 55 60 Ser Leu Ser 65 3 27 DNA Artificial
Sequence Description of Artificial Sequence PCR Primer 3 caggctggga
gccaaatgaa gaaaatg 27 4 30 DNA Artificial Sequence Description of
Artificial Sequence PCR Primer 4 tgataaggaa cgattgacat tagagttgga
30
* * * * *